title: Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation.
abstract: IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD²²⁵ in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp²²⁵ produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.
authors.affiliation: *Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Mei-Hsuan
authors.initials: MH
authors.lastname: Chen
authors.affiliation: †Waisman Center, University of Wisconsin, WI, USA.
authors.forename: Tracy L
authors.initials: TL
authors.lastname: Hagemann
authors.affiliation: ‡School of Biological and Biomedical Sciences, The University of Durham, UK.
authors.forename: Roy A
authors.initials: RA
authors.lastname: Quinlan
authors.forename: Albee
authors.initials: A
authors.lastname: Messing
authors.affiliation: *Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.
authors.forename: Ming-Der
authors.initials: MD
authors.lastname: Perng
mesh_terms: D038261:Alexander Disease / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000473:pathology; D053178:Caspase 6 / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D005904:Glial Fibrillary Acidic Protein / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D059748:Proteolysis / Q000187:drug effects*
chemical_list: D005904:Glial Fibrillary Acidic Protein; D010455:Peptides; D053178:Caspase 6